Blood coagulation promoting product

ABSTRACT

The disclosure pertains to a concentrate for controlling bleeding in hemophilia, free of thrombin, heparin, thromboplastin activity, anticomplement activity, depressor activity, and activated Factor X, and containing coagulation Factors II, VII, IX, and X in non-activated form, having a specific activity of at least about 1.5 Factor IX units per milligram of total protein and a Factor IX: Factor VII ratio of at least about 6:1. In the method of the invention a blood fraction containing coagulation Factors II, VII, IX, and X is applied to an anion exchanger to adsorb the coagulation Factors, which are selectively eluted therefrom using an aqueous sodium chloride-citrate solution of increasing ionic strength. The eluate is treated to reduce its concentration of non-volatile salt and concentrated to yield the above product.

This application is a division, of application Ser. No. 153,396, filedMay 27, 1980 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to and has among its objects the provision ofnovel blood coagulation components and novel methods of making them.Further objects of the invention will be evident from the followingdescription.

2. Description of the Prior Art

There are estimated to be 100,000 cases of congenital hemophilia in theUnited States. Of these, approximately 20,000 are cases of hemophilia B,the blood of such patients being either totally devoid of plasmathromboplastin component or seriously deficient in plasma thromboplastincomponent. The disease therefore exists in varying degrees of severity,requiring therapy anywhere from every week up to once or twice a year.The completely deficient cases require replacement therapy once everyweek; the partially deficient cases require therapy only when bleedingepisodes occur, which may be as seldom as once a year. The bleedingepisodes in congenital, partially-deficient cases are generally causedby a temporarily acquired susceptibility rather than by injury alone.Intravenous injection of a sufficiently large amount of fresh plasma, oran equivalent amount of fresh blood, temporarily corrects the defect ofa deficient subject. The beneficial effect often lasts for two or threeweeks, although the coagulation defect as measured by in-vitro tests onthe patient's blood appears improved for only two or three days. Suchtherapy with fresh plasma or fresh blood is effective but it has severalserious drawbacks: (1) it requires ready availability of a large amountof fresh plasma; (2) requires hospitalization for the administration ofthe plasma; (3) a great many of the patients become sensitized torepeated blood or plasma infusions and ultimately encounter fataltransfusion reactions; (4) at best plasma can only partially alleviatethe deficiency; and (5) prolonged treatment or surgery is not possiblebecause the large amounts of blood or plasma which are required willcause acute and fatal edema.

Several investigators noted that the mixing of blood of certainhemophilic patients would result in the mutual correction of theclotting defect of each blood. Interpretation of these findings waseventually made by Aggeler and co-workers [Proc. Soc. Exptl. Biol. Med.79:692-696 (1952)] and S. G. White et al. [Blood 8:101-124 (1953)].These workers, studying a male patient with a severe hemorrahagicdiathesis associated with a prolonged clotting time which was clinicallyindistinguishable from classic hemophilia, postulated the existence of anew clotting factor. Aggeler et al, recognizing that the new factor wasa precursor of thromboplastin, named it Plasma Thromboplastin Component(PTC). The work was confirmed by Biggs et al. [Brit. Med. J. 2:1378-1382(1952)] in England, who gave it the name Christmas Factor, and bySoulier and Larrieu [New Eng. J. Med. 249:547-553 (1953)] in France, whocalled it Antihemophilic Factor B. This factor is now officiallydesignated Factor IX.

In collaboration with Aggeler et al., the first fractionated PTCpreparation was prepared [Revue d'Hematologie 9:447-453 (1954)]. The PTCwas absorbed on barium sulfate from a solution of Cohn Fraction IV andeluted with 0.34 M sodium citrate. The yields are very small, and thepost-infusion in-vivo activity was only about one-fourth that predictedfrom in-vitro assays by a prothrombin consumption test.

Later, in collaboration with Aggeler et al., a process was developed bywhich PTC was adsorbed onto barium sulfate from EDTA anticoagulatedplasma, eluted with a sodium chloride-sodium citrate buffer, and furtherpurified by cold ethanol fractionation. This preparation was usedclinically with excellent results [Aggeler, P. M. et al. Trans. 6thCongress, Internat. Soc. Hematol. Grune & Stratton, N.Y., pgs. 490-497(1956)]. The process was later published in detail, and it was pointedout that the process was never commercialized because the Albumin andPlasma Protein Fraction obtained as by-products were contaminated withpotentially hazardous levels of barium [Hink, J. H. and Johnson, F. F.,The Hemophilias, CH. 18, page 156, edited by Brinkhous, Univ. of NorthCarolina Press (1964)]. Biggs et al. in England [Brit. J. Haematol.7:349-364 (1961)] and Janiak and Soulier [Thromb. et Diath. Haemorr.8:406-424 (1962)] in France later prepared Factor IX by a similarprocess, substituting tricalcium phosphate for the barium sulfate.However, spontaneous formation of thrombin always occurred and it wasalways necessary to add heparin, both during and after processing toneutralize the potentially dangerous thrombin.

Tullis et al. [New Eng. J. Med. 273:667-674 (1965)] have prepared andstudied a somewhat similar plasma fraction, which they refer to as"prothrombin complex". In the Tullis process, the blood was collectedthrough a calcium-removing ion exchange resin, and the resultingresin-plasma adsorbed onto DEAE cellulose. Elution was accomplished witha sodium phosphate-sodium chloride buffer stabilized with EDTA. Clinicalstudies on the "prothrombin complex" are described in the reference, butother data characterizing the complex are not available.

In U.S. Pat. No. 3,717,708 (hereinafter referred to as '708) there isdisclosed a lyophilized mixture of coagulation components of humanorigin useful in the treatment of patients, who bleed due to acongenital or acquired deficiency of one or more of the coagulationFactors II, VII, IX, X, prepared from human plasma collected in acitrate anticoagulant, free of heparin, free of thrombin, free of theactivated form of Factor X, free of depressor activity, free ofanti-complement activity, and comprising the coagulation componentsFactors II, VII, IX, X in essentially the same proportions as normalhuman plasma and of a potency equivalent to a specific activity of morethan 0.5 clinical unit of each component per milligram of protein.

The product of '708 is produced by a process which comprises the stepsof applying Cohn Supernatant I, Method 6, from unmodified citrated humanplasma onto an ion exchange resin consisting essentially of cross-linkeddextran chains with diethylaminoethyl groups attached by ether linkagesto the glucose units of the polysaccharide chains, and adsorbing thereonsaid coagulation components of the plasma; selectively eluting thecolumn with ammonium bicarbonate solution of a pH of from about 7.3 toabout 8.2 and increasing molarity; separating the eluate fractioncontaining coagulation components; freezing the separated eluatefraction and removing the ammonium bicarbonate therefrom by lyophilizingthe frozen fraction.

A method of producing a blood-coagulation-promoting preparation fromhuman blood plasma is described in U.S. Pat. No. 4,160,025. The productwas termed FEIBA for Factor VIII Inhibitor Bypassing Activity substance.In the production of FEIBA citrated human plasma is treated with awater-insoluble inorganic coagulation-physiologically-surface-activesubstance in the absence of free calcium ions to generate FEIB-activity.After separation from the above substance the supernatant is treatedwith an anion exchanger to adsorb FEIBA and coagulation Factors II, VII,IX, and X. The adsorbed materials are eluted from the exchanger with 3%sodium chloride (0.51 mole per liter) and 0.1% trisodium citratedihydrate (0.0033 mole per liter). The eluate is dialyzed, frozen, andlyophilized.

A concentrate containing Factors II, IX, and X was prepared by Dike etal. (British Journal of Haematology, 1972, Vol. 22, pages 469-490).Blood plasma was diluted to one-third its volume with distilled waterand adsorbed onto DEAE-cellulose in a chromatographic column. The columnwas washed with 0.02 M sodium citrate and then eluted with an aqueoussolution of 0.1 M sodium chloride, 0.01 M-phosphate, and 0.01 M-citrateto give a Factor VII concentrate. Next, the column was eluted with 0.25M sodium chloride, 0.01 M-phosphate, and 0.01 M-citrate to yield aconcentrate containing Factors II, IX, and X.

Middleton et al. in Vox. Sang., 1973, Vol. 24, pages 441-456, describeda therapeutic concentrate of coagulation Factors II, IX, and X fromcitrated, Factor VIII-depleted plasma. After dilution of the plasma toone-third its volume with water for injection, the plasma was contactedwith a DEAE-cellulose column to adsorb the coagulation Factors. Then,the cellulose was washed with an aqueous solution containing 0.03M-phosphate and 0.03 M-citrate and eluted with an aqueous solutioncontaining 0.03 M-phosphate, 0.03 M-citrate, and 0.2 M sodium chlorideto give a concentrate of Factors II, IX, and X. This concentratecontained thrombin in amounts greater than the limits set by the U.S.National Institutes of Health. The authors note that the presence ofphosphate is required in the eluting medium.

In Vox. Sang., 1977, Vol. 33, pages 37-50, Suomela et al. described amethod for large-scale preparation of a coagulation Factor IXconcentrate. Coagulation Factors II, VII, IX and X from human plasma areadsorbed onto DEAE Sephadex®. The gel was washed with an aqueous buffersolution containing 0.16 M sodium chloride and 0.04 M-phosphate and theneluted with a mixture of 0.5 M and 1.0 M phosphate buffer to give aFactor IX concentrate. The Factor IX:Factor VII ratio of this productwas about 2:1 to 3:1 and the Factor IX:NAPTT ratio was about 3:1. Thethrombin activity of the above concentrate was lower than 0.001 Unitsper milliliter.

SUMMARY OF THE INVENTION

I have discovered novel methods for producing blood coagulationcomplexes comprising coagulation Factors II, VII, IX, and X (hereinafterreferred to as "Factor IX concentrates"). Surprisingly, the novel FactorIX concentrates produced by the method of my invention possess at leastabout 50% more activity than the '708 products.

In the method of the invention a blood plasma fraction containingcoagulation Factors II, VII, IX, and X in non-activated form is appliedto an anion (basic ion) exchanger to adsorb the coagulation Factors.Next, the exchanger is washed with an aqueous solution containing anon-volatile salt and citrate ions and having an ionic strengthsufficient to remove less strongly bound blood plasma proteins from theexchanger but insufficient to remove the adsorbed coagulation Factorsfrom the exchanger. Then, the exchanger is treated with an aqueoussolution containing a non-volatile salt and citrate ion and having anionic strength sufficient to elute the adsorbed coagulation Factors butinsufficient to remove these blood plasma proteins more strongly boundto the anion exchanger.

The eluate is treated to reduce its concentration of non-volatile saltand concentrated to yield the product of the invention.

The combined wash step and elution step, both with an aqueous solutioncontaining a non-volatile salt and citrate ions, may be viewed as aselective elution of the exchanger by increasing the ionic strength ofthe aforementioned aqueous solution in the absence of phosphate ions.

It is important that blood coagulation complexes or concentrates do notcontain thrombin since the injection of thrombin into a human would beconsidered highly dangerous. Although it is possible to neutralizethrombin activity with heparin, it would be preferable not to have thethrombin there in the first place. Heparin is undesirable in a Factor IXcontaining concentrate because it also is potentially hazardous to thepatient and because it causes difficulties in assaying the coagulationfactors in the concentrate. In the administration of a Factor IXcontaining concentrate, constant monitoring of the patient's coagulationstatus is required, and the presence of heparin will not only complicatethis testing procedure, but will make the results thus obtainedunreliable. An advantage of this invention is that the concentrate befree of both thrombin and of heparin.

Another advantage of this invention is the provision of a concentratefree of the anticomplement activity which is so often associated withplasma globulins and which would make the intravenous administration ofthe product highly dangerous.

An important advantage of the invention is that the product thereof hassubstantially reduced thrombogenicity in comparison to the prior artconcentrates.

A further advantage of the product of the invention is that the ratio ofFactor IX to Factor VII is substantially greater than that for the '708product. Thus, the present Factor IX concentrate contains less FactorVII than does the '708 material. The present product, therefore, has adecreased ability to contribute to hemostasis.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The product of the invention is prepared from human blood plasma. Thepreponderance of blood generally taken and available for transfusion isprotected from coagulation by treatment with a citrate anticoagulantwhich allows the blood to be utilized for only a limited period of time.After this limited time expires, this blood must be discarded or it canbe made available for fractionation into certain useful components. As amatter of fact, the major source of whole blood converted to plasma forfractionation comes from blood collected by plasmapheresis, which hasbeen protected by citrate anticoagulation. It is, therefore, importantthat processes for the fractionation of plasma, such as to obtain aFactor IX containing concentrate, be directed to citrate preservedblood, to which the instant method is.

As mentioned above, in the first step in the process of the invention ahuman blood plasma fraction containing coagulation Factors II, VII, IX,and X is contacted with a basic ion exchanger on which the coagulationFactors are adsorbed. A number of fractionation methods have beenapplied to human blood plasma, and these methods are summarized in "ThePlasma Proteins:", second edition, Vol. III, pages 548-550, AcademicPress, New York, New York (1977). The preferred starting material in theprocess of the invention is Effluent I (Cohn Supernatant I) from a Cohnfractionation method (U.S. Pat. No. 2,390,074 and J. Am. Chem. Soc.,Vol. 68, page 459 (1946). Effluent I is contacted with an anionic orbasic ion exchanger, for example, DEAE (diethylaminoethyl) Sephadex®consisting of cross-linked dextran chains with diethylaminoethyl groupsattached by ether linkages to the glucose units of the polysaccharidechains, supplied by Pharmacia Fine Chemicals, Inc., Piscataway, NewMarket, N.J., to adsorb the aforementioned coagulation Factors. Thespent Effluent I is returned to the plasma fractionation process so thatnone of the other plasma components is wasted.

Generally, contact between Effluent I and DEAE Sephadex is achieved byforming a bed of freshly equilibrated DEAE Sephadex and passing EffluentI therethrough. Approximately 10 g. (wet weight) of DEAE Sephadex perliter of Effluent I is used although the amounts employed may be variedover a wide range.

The anion exchanger is washed next to remove those plasma proteins,including ceruloplasmin, that are less strongly bound to the exchangerthan the coagulation Factors. The wash solution should contain anon-volatile salt and have an ionic strength sufficient to remove theseless strongly bound plasma proteins but insufficient to remove theadsorbed coagulation Factors. The ionic strength (I) of the washsolution should be about 0.10-0.45. A suitable non-volatile salt issodium chloride in a concentration of 0.1-0.3 M. It is necessary thatthis aqueous sodium chloride solution contain citrate ions (sodiumcitrate or the like) at a concentration of about 0.05-0.2 M. A preferredwash solution in accordance with the invention is 0.2 M sodium chloridecontaining 0.1 M sodium citrate (I=0.26). Other aqueous salt solutionsmay be employed with the proviso that the salt be non-volatile andphysiologically compatible with the final product. Generally, the pH ofthe aqueous solution should be about 6 to 9, preferably, about 7 to 8.

Prior to this wash step it may be desired to wash the anion exchangerspecifically to remove ceruloplasmin. To this end the exchanger can bewashed with an aqueous solution of a volatile salt such as ammoniumbicarbonate, about 0.1-0.3 M. Generally, the concentration of volatilesalt should be sufficient to remove the ceruloplasmin but insufficientto remove the adsorbed coagulation Factors.

The anion exchanger, having been washed as described above, is treatedwith an aqueous solution containing a non-volatile salt and citrate ionsand having an ionic strength sufficient to elute the adsorbedcoagulation Factors but insufficient to remove those plasma proteinsmore strongly bound to the exchanger. For this purpose the ionicstrength of the aqueous solution should be greater than 0.50, preferably0.81, and within the range of about 0.50-2.5. Thus, the solution shouldcontain 0.35-2.0 M non-volatile salt and 0.005-0.02 M citrate ions. Itis preferred that the eluting solution contain the same non-volatilesalt as the aforementioned wash solution. A preferred eluting solution,then, is 0.35-2.0 M aqueous sodium chloride containing 0.005-0.02 Mcitrate ions. The pH of the eluting solution should be about 6 to 9,preferably about 7 to 8.

The eluate is treated to reduce its water content and the concentrationof non-volatile salt, i.e., reduce the concentration of salt in theeluate, to a physiologically acceptable level, i.e., to 0.15 m or less.The aforementioned objective may be accomplished, for example, by usinga combination of ultrafiltration and diafiltration as is known in theart. It is preferred that the ultrafiltration and diafiltration becarried out against an aqueous sodium chloride-sodium citrate buffer infinal container concentration, i.e., the concentration of the above asfound in the final container. Generally, final container concentrationof sodium chloride is 0.088 M and of sodium citrate, 0.05 M. Other meansof achieving removal of the salt will be suggested to those skilled inthe art. It is to be noted that reduction of the amount of water may beaccomplished by other methods with the exception of lyophilization,which is to be avoided.

The Factor IX concentrates can be formulated into pharmaceuticalpreparations for therapeutic, diagnostic, or other uses. To prepare themfor intravenous administration the compositions are dissolved usually inwater containing physiologically compatible substances such as sodiumchloride, glycine, and the like and having a buffered pH compatible withphysiological conditions. Generally, guidelines for intravenouslyadministered compositions are established by governmental regulations.

The product prepared in accordance with the above method ischaracterized as substantially free of thrombogenicity and free ofthrombin, heparin, thromboplastin activity, anticomplement activity,depressor activity, and activated Factor X, comprising coagulationFactors II, VII, IX and X. It has a specific activity of at least about1.5 Factor IX units per mg. total protein. One Factor IX unit is definedas that amount of Factor IX activity contained in one ml. of freshaverage pooled plasma. The Factor XI:Factor VII ratio is at least about6:1 or more, preferably 9:1 or more. The Factor IX:Factor II ratio andthe Factor IX:Factor X ratio are about 1:1 to 2:1. Factor IX:NAPTT isabout 5:1 or greater.

A modification of the product of this invention is the product in anelectrolyte buffer suitable for intravenous administration as follows:

The ultrafiltered/diafiltered eluate is sterilized by filtration througha sterile 0.2 micron porosity membrane filter and aseptically filled inconvenient portions into sterile bottles, frozen and lyophilized andstored at 5° C.

For human administration, the content of the vial is asepticallyredissolved in water for injection. Alternatively, the product can besterile filled and lyophilized to a water-free product. In this instanceit is reconstituted with a suitable isotonic diluent prior to humaninjection.

The above process recites the details which are preferred at this time.However, many variations can be made without departing from the generalprinciples of the invention.

For example, the amount of DEAE Sephadex relative to the volume ofEffluent I may be varied over a wide range. The use of larger amounts ofDEAE Sephadex will result in a lower degree of purity and greater yieldof the coagulation complex, and a greater loss of other plasma proteinsnormally recoverable in subsequent steps. The use of smaller amounts ofDEAE Sephadex will result in even higher degrees of purity, but willalso result in considerably lower yields. Although DEAE Sephadex is thepreferred anionic exchanger, presumably other anionic adsorbents wouldwork also.

The elution step with an aqueous solution containing a non-volatile saltand citrate ions is extremely important to the process of thisinvention. Only in this fashion is there obtained a product having atleast about 50% more activity than the '708 product. Furthermore, it isonly in this manner that a less thrombogenic product can be secured.

Another aspect of the present invention concerns an improvement in the'708 process wherein a combined ultrafiltration-diafiltration is used inplace of lyophilization to remove ammonium bicarbonate. It has beenfound that the specific activity of the product prepared by the improvedmethod of the invention is greater than that of the '708 product and,more important, the new product is substantially free ofthrombogenicity. The advantages of the present invention can be obtainedby applying a combination of ultrafiltration and diafiltration to the'708 eluate after its separation from the anion exchanger in order toremove ammonium bicarbonate and water therefrom. The eluate is subjectedfirst to diafiltration to remove substantially all the ammoniumbicarbonate. The eluate, therefore, is contacted with a semi-permeablemembrane until the ammonium bicarbonate level is reduced to less thanabout 0.01 M. Membranes suitable for the diafiltration should have anominal molecular weight cut-off of about 10,000-80,000 daltons. Typicalmembranes suitable for this step of the process of the invention areAmicon PM10 and Amicon H10 P10 (Amicon Corp., Lexington, Mass.),Millipore PTGC (Millipore Corp., Bedford, Mass.), and Romicon PM10 andRomicon GM80 (Romicon Corp., Wicoburn, Mass.).

Following the diafiltration the eluate is subjected to ultrafiltrationby contacting it with a suitable semi-permeable membrane until thedesired level of water removal has been obtained. Membranes suitable forthis step of the process of the invention should have a nominalmolecular weight cut-off of about 10,000-80,000 daltons. Typicalmembranes suitable for the ultrafiltration step are those enumeratedabove for the diafiltration procedure.

In a preferred embodiment of the invention ultrafiltration of the eluateis conducted with hollow fibers in an ultrafiltration unit such as, forexample, the Amicon DC30 (30 sq. ft. filtration area/hollow fiber unit)using an ultrafiltration membrane such as the Amicon PM10 (10,000daltons molecular weight cut-off) or the equivalent.

After ultrafiltration the material is adjusted for pH, sterile filtered,sterile filled and lyophilized.

EXAMPLES

The invention is demonstrated further by the following illustrativeexamples.

ASSAY METHODS

Factors II and VII: Factor II and Factor VII were assayed by the methodof Owren described in the Scand. J. Clin. and Lab. Investigation, Vol.1, page 81 (1949).

Factors X and Xa: Factor X and Factor Xa were assayed by the method ofBachmen et al, described in Thromb. Diath. Haemorrh., Vol. 2, page 24,(1958).

Thrombin: The assay procedure employed was described by Fenton II et al,in Thrombosis Res., Vol. 4, pages 809-817 (1974).

Factors IX and VIII: Modification of the procedures described byLangdell et al (partial thromboplastin time technique), J. Lab. Clin.Med., Vol. 41, pages 637-647 (1953) and by Proctor et al (kaolinclotting time method) Amer. J. Clin. Path., Vol. 36, page 212 (1961)were employed. Platelet Factor 3 was supplied by a cephalin suspension.Maximum surface contact activation was achieved with Celite® powder. Allother clotting factors (except Factor IX or Factor VIII) were suppliedby a substrate comprising plasma from a patient severely deficient inFactor IX or Factor VIII mixed with barium sulfate adsorbed beef plasma.Quantitation of an unknown specimen was made by comparing its clottingtime in the test with that achieved by dilutions of a normal standard.

The exact assay procedure is the same for both Factor IX and Factor VIIIexcept that the activator in Factor IX assay is Platelin® Plus Activatorinstead of automated APTT reagent (General Diagnotics, Inc., MorrisPlains, N.J.).

Non-Activated Partial Thromboplastin Time (NAPTT):

A 0.1-ml. sample of NAPTT Substrate Plasma (stored on ice), a 0.1-ml.sample of Partial Thromboplastin with no activator (stored on ice), and0.1-ml. of the sample to be tested (in various dilutions) were added toa 10×75 mm polystyrene test tube, mixed by shaking carefully and placedin 37° C. water bath with simultaneous starting of a stopwatch. Afterexactly a one-minute period, 0.1 ml 0.025 M CaCl₂ maintained at 37° C.was added with simultaneous starting of the timer and the contents ofthe tube were mixed by careful shaking. Thereafter, the tube was heldundisturbed for 30 seconds to 1 minute, depending upon the type ofsample. It was then tilted from time to time until gelation started or aclot was observed with care being taken to minimize exposure to roomtemperature which was below 37° C. Time was recorded at the first signof a clot formation. Development of opacity and gelation preceded theformation of a firm clot.

When the sample was replaced by Tris buffer, NAPTT blank time wasobtained. If the blank time was over 300 seconds, the assay wascontinued. Any substrate plasma which gave a blank time of less than 300seconds was unacceptable for this test.

By obtaining NAPTT times at a wide range of dilutions and plotting timesin seconds on ordinate and dilutions on abscissa, a straight linerelation was obtained. In most prothrombin complex concentrates,inhibitory effect was observed at low dilutions. Therefore, a straightline relationship was observed at 1:100 and higher dilutions. By using asuitable standard such as Factor IX-1 (FN-1) of the Bureau of Biologicsand assigning 100 units per ml for this standard, it was possible toconstruct a standard curve. On this standard curve, the NAPTT times of agiven sample were read and expressed as units. Longer NAPTT times (closeto blank time) expressed as low NAPTT units/ml indicate reducedthrombogenicity.

EXAMPLE 1

Effluent I (1500 liters) was contacted with 15 kg. of DEAE Sephadex geland mixed together. After two hours the mixture was filtered to give 15kg. of gel which was washed sequentially with 50 liters of 0.2 Mammonium bicarbonate, 50 liters of 0.3 M ammonium bicarbonate and 45liters of 0.01 M sodium citrate-0.2 M sodium chloride (pH 7.1) buffer.Elution was carried out with 33.7 liters of 0.01 M sodium citrate-0.75 Msodium chloride at pH 7.24 to obtain a protein solution of A₂₈₀ =10.2.This was concentrated by ultrafiltration to A₂₈₀ of about 38.2 anddiafiltered to 5 volume exhange against 0.05 M sodium citrate-0.088 Msodium chloride (pH 7.19) buffer.

The above material was sterile filtered, sterile filled in vials (20 ml.fill volume) and freeze dried. The contents of the vials were assayed bythe aforementioned methods. The results are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Coagulation Factors (U/ml)                                                                    Specific activity                                                                          NAPTT                                            II   VII     IX      X    (U/mg).sup.a                                                                             (sec.).sup.b                                                                        (U/ml)                             ______________________________________                                        29   4.3     51.7    36   2.21       248   7.2                                ______________________________________                                         .sup.a Units IX per mg. of total                                              .sup.b 1:100 dilution                                                    

EXAMPLE 2

Effluent I (1300 liters) was contacted with 13 kg. of DEAE Sephadex geland mixed together. After two hours the mixture was filtered to give 13kg. of gel which was washed sequentially with 50 liters of 0.2 Mammonium bicarbonate, 55 liters of 0.3 M ammonium bicarbonate and 45liters of 0.01 M sodium citrate-0.2 M sodium chloride (pH 7.18) buffer.Elution was carried out with 36.5 liters of 0.01 M sodium citrate-0.75 Msodium chloride (pH 7.16) to obtain a protein solution of A₂₈₀ =9.65.This was concentrated by ultrafiltration to A₂₈₀ =29.3 and diafilteredto 5 volume replacement against 0.05 M sodium citrate-0.088 M sodiumchloride (pH 7.16).

The above material was sterile filtered, sterile filled (20 ml. fillvolume) and freeze dried. The contents of the vials were assayed by theaforementioned methods. The results are outlined in Table 2.

                  TABLE 2                                                         ______________________________________                                        Coagulation Factors (U/ml)                                                                    Specific Activity                                                                          NAPTT                                            II   VII     IX      X    (U/mg)     (sec.).sup.a                                                                        (U/ml)                             ______________________________________                                        24   5.4     37      39   1.53       272   5.8                                ______________________________________                                         .sup.a 1:100 dilution                                                    

EXAMPLE 3

Table 3 contains a side-by-side comparison of the products of Examples 1and 2 and products prepared by the process of '708 (as described atcolumns 3 and 4).

                  TABLE 3                                                         ______________________________________                                                IX      Specific Activity                                                                          NAPTT                                            Product (U/ml)  (IX/mg protein)                                                                            (U/ml) IX/NAPTT                                  ______________________________________                                        Invention.sup.a                                                                       44.4    1.9          6.8    6.54                                      '708.sup.b                                                                            27.1    1.1          8.9    3.30                                      ______________________________________                                         .sup.a Average of 2 runs                                                      .sup.b Average of 5 runs                                                 

EXAMPLE 4

A comparison of the concentrations of the coagulation Factors in theproduct of the invention prepared in Examples 1 and 2 and the product of'708 is outlined in Table 4.

                  TABLE 4                                                         ______________________________________                                                Coagulation Factors (U/ml)                                            Product   II      VII     IX    X     IX/VII                                  ______________________________________                                        Invention.sup.a                                                                         26.5    4.9     44.4  37.5  9.44                                    '708.sup.a                                                                              18.3    9.5     26.1  29.3  2.80                                    ______________________________________                                         .sup.a Average of 2 runs                                                 

EXAMPLE 5

The procedure of Example 2 was employed to prepare a product inaccordance with the invention. The so-prepared product was assayed bythe aforementioned methods and then held for a period of 8 months todetermine its in vitro stability. The so-held product was assayed asabove and the results are outlined in Table 5.

                  TABLE 5                                                         ______________________________________                                                               NAPTT                                                          Coagulation Factors (U/ml)                                                                     1:10.sup.a                                                                            1:100.sup.a                                  Product held                                                                            II       IX       X      (sec.)                                                                              (sec.)                               ______________________________________                                        0 months  24.2     38.7     37.5   161   259                                  8 months  21.8     40.2     40.8   172   277                                  ______________________________________                                         .sup.a Dilution                                                          

EXAMPLE 6

The procedure described in '708 columns 3 and 4 was followed to Step Dto yield an eluate, which was divided into two equal parts. As a controlone part was lyophilized and the powder was dissolved in 0.088 M sodiumchloride-0.05 M sodium citrate at pH 6.9. The other part of the eluatewas diafiltered to 5 volume replacements against the same buffer using10,000 daltons cut-off hollow fiber membrane (Amicon PM10) followed byultrafiltration for concentration. Five different starting lots (1-5)were investigated, and the results are summarized in Table 6.

                  TABLE 6                                                         ______________________________________                                        Diafiltration plus Ultrafiltration                                                               Control                                                         IX     Specific  NAPTT.sup.a                                                                          IX   Specific                                                                              NAPTT.sup.a                         Lot  (U/    Activity  (secs) (U/  Activity                                                                              (secs)                              No.  ml)    (IX/A.sub.280)                                                                          (1/100)                                                                              ml)  (IX/A.sub.280)                                                                        (1/100)                             ______________________________________                                        1    27.6   1.08      360    24.6 0.94    201                                 2    24.9   1.07      290    14.4 0.94    240                                 3    24.1   1.11      370    21.6 0.92    208                                 4    30.8   1.10      340    27.6 0.70    152                                 5    34.8   1.01      200    26.8 0.65    142                                 ______________________________________                                    

What is claimed is:
 1. A process for the production of a bloodcoagulation complex containing coagulation Factors II, VII, IX, and X innon-activated form, which comprises the steps of(a) applying a bloodplasma fraction containing coagulation Factors II, VII, IX, and X to ananion exchanger to adsorb the coagulation Factors, (b) washing theexchanger with an aqueous solution containing a non-volatile salt andcitrate ions and having an ionic strength sufficient to remove lessstrongly bound blood plasma proteins from the exchanger but insufficientto remove the adsorbed coagulation Factors, (c) eluting the adsorbedcoagulation Factors from the exchanger with an aqueous solutioncontaining a non-volatile salt and citrate ions and having an ionicstrength sufficient to remove the coagulation Factors, but insufficientto remove blood plasma proteins more strongly bound to the exchanger,and (d) treating the eluate to reduce the concentration of non-volatilesalt to a physiologically acceptable level.
 2. The method of claim 1which further includes the step of washing the exchanger prior to Step(b) with an aqueous solution of a volatile salt having a concentrationsufficient to remove adsorbed ceruloplasmin but insufficient to removethe adsorbed coagulation Factors.
 3. The method of claim 2 wherein thevolatile salt is ammonium bicarbonate.
 4. The method of claim 3 whereinthe concentration of ammonium bicarbonate in the aqueous solution isabout 0.1-0.3 mole per liter.
 5. The method of claim 1 wherein the ionicstrength of the aqueous solution of Step (b) is about 0.1-0.45.
 6. Themethod of claim 1 wherein the ionic strength of the aqueous solution ofStep (c) is about 0.5-2.5.
 7. The method of claim 1 wherein the eluateof Step (d) is subjected to diafiltration to remove non-volatile salt.8. The method of claim 1 wherein ceruloplasmin is washed from theexchanger in Step (b).
 9. A blood coagulation complex containingcoagulation Factors II, VII, IX, and X in non-activated form prepared bythe process of claim
 1. 10. A lyophilized stable composition forcontrolling bleeding in hemophilia containing coagulation Factors II,VII, IX and X in non-activated form prepared by a process whichcomprises the steps of(a) applying a blood plasma fraction containingcoagulation Factors II, VII, IX, and X to an anion exchanger to adsorbthe coagulation Factors, (b) selectively eluting the exchanger with anaqueous solution containing a non-volatile salt and citrate ions ofincreasing ionic strength, (c) separating an eluate containing thecoagulation Factors, (d) treating the eluate to reduce the volatile saltconcentration to a physiologically acceptable level, and (e)lyophilizing the eluate.
 11. The composition of claim 10 wherein theprocess further includes the step of subjecting the eluate of Step (d)to sterile filtration.
 12. A pharmaceutical preparation comprising thecomposition of claim
 10. 13. The composition of claim 10 wherein theeluate is subjected to diafiltration to remove the non-volatile salt.14. In a process for the production of a blood coagulation complexcontaining coagulation Factors II, VII, IX, and X in non-activated form,which process comprises the steps of applying a blood plasma fractioncontaining coagulation Factors II, VII, IX, and X to an anion exchangerto adsorb the coagulation Factors, selectively eluting the exchangerwith a volatile salt solution of increasing molarity, and removing thevolatile salt from the eluate, the improvement which comprisessubjecting the eluate to diafiltration to remove substantially all thevolatile salt and then subjecting the eluate to ultrafiltration toremove water therefrom.
 15. The process of claim 14 wherein the volatilesalt is ammonium bicarbonate.
 16. A composition containing coagulationFactors II, VII, IX, and X in non-activated form prepared by the processof claim 14.